Dna sequences, recombinant dna molecules and processes for producing pi-linked lymphocyte function associated antigen-3

ABSTRACT

Polypeptides that bind to CD2, the receptor on the surface of T-lymphocytes. Most preferably, the polypeptides are soluble. DNA sequences that code on expression and/or secretion in appropriate unicellular hosts for those polypeptides.

This invention relates to DNA sequences, recombinant DNA molecules andprocesses for producing Lymphocyte Function Associated Antigen-3(PI-Linked form of LFA-3). More particularly, the invention relates toDNA sequences that are characterized in that they code on expression inan appropriate unicellular host for a soluble PI-linked form of LFA-3 orderivatives thereof that bind to CD2, the receptor on the surface ofT-lymphocytes. In accordance with this invention, unicellular hoststransformed with these DNA sequences and recombinant DNA moleculescontaining them may also be employed to produce LFA-3 essentially freeof other proteins of human origin. This novel antigen may then be usedin the therapeutic and diagnostic compositions and methods of thisinvention.

BACKGROUND OF THE INVENTION

T-lymphocytes play a major role in the immune response by interactingwith target and antigen presenting cells. For example, the T-lymphocytemediated killing of target cells is a multi-step process involvingadhesion of a cytolytic T-lymphocyte to a target cell. And, helperT-lymphocytes initiate the immune response by adhesion toantigen-presenting cells.

These interactions of T-lymphocytes with target and antigen-presentingcells are highly specific and depend on the recognition of an antigen onthe target or antigen-presenting cell by one of the many specificantigen receptors on the surface of the T-lymphocyte.

The receptor-antigen interaction of T-lymphocytes and other cells isalso facilitated by various T-lymphocyte surface proteins, e.g., theantigen receptor complex CD3(T3) and accessory molecules CD4, LFA-1,CD8, and CD2. It is also dependent on accessory molecules, such asLFA-3, ICAM-1 and MHC that are expressed on the surface of the target orantigen-presenting cells. In fact, it is hypothesized that the accessorymolecules on the T-lymphocytes and on the target or antigen-presentingcells interact with each other to mediate intercellular adhesion.Accordingly, these accessory molecules are thought to enhance theefficiency of lymphocyte-antigen-presenting cell and lymphocyte-targetcell interactions and to be important in leukocyte-endo-thelial cellinteractions and lymphocyte recirculation.

For example, recent studies have suggested that there is a specificinteraction between CD2 (a T-lymphocyte accessory molecule) and LFA-3 (atarget cell accessory molecule) that mediates T-lymphocyte adhesion tothe target cell. This adhesion is essential to the initiation of theT-lymphocyte functional response (M. L. Dustin et al., "PurifiedLymphocyte Function-Associated Antigen-3 Binds To CD2 And Mediates TLymphocyte Adhesion", J. Exp. Med., 165, pp. 677-92 (1987); Springer etal., "The Lymphocyte Function-Associated LFA-1, CD2, and LFA-3Molecules: Cell Adhesion Receptors Of The Immune System", Ann. Rev.Immunol., 5, pp. 223-52 (1987)). And, monoclonal antibodies to eitherLFA-3 or CD2 have been shown to inhibit a spectrum of cytolytic Tlymphocyte and helper T lymphocyte dependent responses (F.Sanchez-Madrid et al., "Three Distinct Antigens Associated With HumanT-Lymphocyte-Mediated Cytolysis: LFA-1, LFA-2, And LFA-3", Proc. Natl.Acad. Sci. USA, 79, pp. 7489-93 (1982)).

LFA-3 is found on antigen-presenting cells, and target cells,specifically on monocytes, granulocytes, CTL's, B-lymphoblastoid cells,smooth muscle cells, vascular endothelial cells, and fibroblasts(Springer et al., supra). LFA-3 exists as two distinct cell surfaceforms (Dustin et al., "Anchoring Mechanisms For LFA-3 Cell AdhesionGlycoprotein At Membrane Surface", Nature, 329, pp. 846-848 (1987)).These forms differ mainly by their mechanism of attachment to lipidbilayers of biological membranes. One such anchoring mechanism is via astretch of hydrophobic amino acids, also referred to as a transmembranedomain, which penetrates the lipid bilayers. cDNA encoding this form ofLFA-3, also referred to as an integrated membrane form, has been clonedand sequenced (B. Wallner et al., J.Exp.Med., 166, pp. 923-32 (1987)).

Alternatively, LFA-3 has been reported to insert into the membrane ofB-lymphoblastoid cells via a phosphatidylinositol ("PI")-containingglycolipid covalently attached to the C-terminus of the protein (Dustinet al., supra). Membrane insertion of this type was deduced by observingthe presence of protein after adding to the cell surfacephosphatidylinositol-specific phospholipase. This enzyme releases onlythe PI-linked form of proteins. It does not affect the integratedmembrane form. Thus, the release of LFA-3 in the presence of this enzymesuggests that LFA-3 has a PI-linked form.

The PI-linked form of LFA-3 is believed to be derived from alternativeRNA splicing of a gene transcript. It appears to be selectivelyexpressed in different cell types, and during different stages ofdevelopment than the transmembrane form of LFA-3.

It would be desirable to obtain large amounts of a recombinant PI-linkedform of LFA-3, than would be available from purification from naturalsources, e.g. lymphoblastoid cells. More desirable would be to obtainlarge amounts of soluble LFA-3 from a PI-linked form of LFA-3.

SUMMARY OF THE INVENTION

This invention solves these problems. One aspect of this invention isthe production of a recombinant PI-linked form of LFA-3. Another aspectof this invention is the production of soluble LFA-3 from a PI-linkedform of LFA-3. The latter embodiment is accomplished by expressing DNAsequences encoding a PI-linked form of LFA-3 in cell lines deficient ina PI-linkage attachment mechanism. A still further aspect of thisinvention is the process of producing a soluble LFA-3 derived from aPI-linked form of LFA-3. This embodiment is accomplished by removingthose portions of the DNA sequence encoding the hydrophobictransmembrane region of the PI-linked form of LFA-3.

This invention accomplishes each of these goals by providing DNAsequences coding on expression in an appropriate unicellular host for aPI-linked form of LFA-3 or derivatives thereof.

This invention also provides recombinant DNA molecules containing thoseDNA sequences and unicellular hosts transformed with them. Those hostspermit the production of large quantities of the PI-linked form ofLFA-3, and its derivatives, of this invention for use in a wide varietyof therapeutic and diagnostic compositions and methods.

The DNA sequences of this invention are selected from the groupconsisting of:

(a) the DNA sequence of the DNA insert carried in phage P24; and

(b) DNA sequences which code on expression for a polypeptide coded foron expression by the foregoing DNA sequence.

The DNA sequences of this invention are also selected from derivativesof the DNA insert carried in phage λP24 produced by removing thoseportions of the DNA sequence encoding the hydrophobic transmembraneregion of the PI-linked form of LFA-3.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the amino acid sequences of the N-terminal and variouspeptide fragments of human LFA-3, purified from human erythrocytes usingimmunoaffinity chromatography.

FIG. 2 depicts two pools of chemically synthesized oligonucleotide DNAprobes derived from the amino acid sequence of a human LFA-3 purifiedfrom human erythrocytes.

FIG. 3 depicts the DNA sequence of the DNA insert carried in phage P24and the amino acid sequence deduced therefrom.

FIG. 4 depicts the relevant portions of sequencing plasmid pNNOl.

FIG. 5 depicts the nucleotide sequence of probes LF-10, LF-11, NN-A,NN-B, NN-C, and NN-D.

FIGS. 6A and 6B together depict a comparison of the DNA insert carriedin phage P24 (and the deduced amino acid sequence) and the DNA insertcarried in phage λHT16 (and the deduced amino acid sequence), whichcodes for an integrated membrane form of LFA-3.

DESCRIPTION OF THE INVENTION

We isolated the DNA sequences of this invention from a λgtl0 cDNAlibrary derived from peripheral blood lymphocytes. However, we couldalso have employed libraries prepared from other cells that express aPI-linked form of LFA-3. These include, for example, monocytes,granulocytes, CTL's, B-lymphoblastoid cells, smooth muscle cells,endothelial cells and fibroblasts. We also could have used a humangenomic bank.

For screening this library, we used a series of chemically synthesizedanti-sense oligonucleotide DNA probes. We selected these probes from aconsideration of the amino acid sequences of various fragments of LFA-3that we determined using LFA-3 purified from human erythrocytes. Thesefragments are depicted in FIG. 1. We selected amino acids from variousareas that permitted the construction of oligonucleotide probes ofminimal degeneracy.

We prepared two pools of probes: LFl and LF2-5. These pools are depictedin FIG. 2. LFl is a 32-fold degenerate 20-mer and LF2-5 is a 384-folddegenerate 20-mer. Because of the high degeneracy of this latter pool,we subdivided the pool into four subpools --LF2, LF3, LF4 and LF5 --of96-fold degeneracy each.

For screening, we hybridized our oligonucleotide probes to our cDNAlibraries utilizing a plaque hybridization screening assay. We selecteda clone --P24 --hybridizing to one of our probes. And, after isolatingand subcloning the cDNA insert of the selected clone, P24, intoplasmids, we determined its nucleotide sequence and deduced the aminoacid sequence from that nucleotide sequences.

We have depicted in FIG. 3 the nucleotide sequence of the cDNA insert ofphage λP24 and the amino acid sequence deduced therefrom. As shown inFIG. 3, this cDNA insert has an open reading frame of 720 bp (240 aminoacids), a 17 bp 5' untranslated region and a 93 bp 3' untranslatedregion. Also present is a transmembrane domain, i.e., from N₆₆₂ -N₇₂₅.The 3' untranslated region of P24 contains a poly(A) adenylation site.The P24 cDNA codes for a 240 amino acid protein, including a 28 aminoacid signal sequence.

We have depicted in FIGS. 6A and 6B a comparison of the DNA sequencesand deduced amino acid sequences of a membrane integrated form of LFA-3(HT16) and the PI-linked form of LFA-3 of this invention. From thiscomparison, it is apparent that the last 14 amino acids (AA₂₀₉ -AA₂₂₂)including a cytoplasmic domain at the C-terminus of the membraneintegrated form of LFA-3 are replaced by 4 different amino acids in thePI-linked form of LFA-3.

The DNA sequences of this invention are selected from the groupconsisting of:

(a) the DNA sequence of the DNA insert

P24 carried in phage P24; and

(b) DNA sequences which code on expression for a polypeptide coded foron expression by the foregoing DNA sequence,

e.g., the cDNA sequence depicted in FIG. 3 and contained in depositedclone λP24, may be used, as described below, in a variety of ways inaccordance with this invention.

The DNA sequences, portions of them, or synthetic or semi-syntheticcopies of them, may be used as a starting material to prepare variousmutations. Such mutations may be either silent, i.e., the mutation doesnot change the amino acid sequence encoded by the mutated codon, ornon-silent, i.e., the mutation changes the amino acid sequence encodedby the mutated codon. Both types of mutations may be advantageous inproducing or using the LFA-3's of this invention. For example, thesemutations may permit higher levels of production, easier purification,or production of secreted shortened or soluble forms of PI-linked formsof LFA-3.

The DNA sequences of this invention are also useful for producing thePI-linked forms of LFA-3, or its derivatives, coded on expression bythem in unicellular hosts capable of attaching proteins by PI-linkage,e.g., CHO cells, transformed with those DNA sequences. Preferably,according to a second embodiment of this invention, these DNA sequencesmay be expressed in a cell line deficient in the PI-linkage attachmentmechanism, such as mouse L-cells.e.g., L-M (tk⁻) cells. In this case theLFA-3 may be secreted into the medium in a soluble form. This secretedform of LFA-3 is approximately 3 kd smaller than other forms of LFA-3retained intracellularly in L-M (tk⁻) cells or extracted from CHO cells.While not wishing to be bound by theory, we believe that the DNAsequences of the present invention produce and secrete a smaller solubleform of LFA-3 because a portion of the transmembrane region is cleavedbefore or after secretion by cells deficient in a PI-linkage attachmentmechanism, and therefore efficient attachment of the PI-linked form ofLFA-3 to the cell surface is prevented.

According to another embodiment of this invention DNA sequences encodinga PI-linked form of LFA-3 may be modified as compared to that of FIG. 3(amino acids -28 to 212) to remove from it portions that code for thehydrophobic transmembrane region, e.g., from about nucleotide 662 to731, to allow production of soluble LFA-3 protein in any celltransformed with those modified sequences.

As well known in the art, the DNA sequences of this invention areexpressed by operatively linking them to an expression control sequencein an appropriate expression vector and employed in that expressionvector to transform an appropriate unicellular host.

Such operative linking of a DNA sequence of this invention to anexpression control sequence, of course, includes the provision of atranslation start signal in the correct reading frame upstream of theDNA sequence. If the particular DNA sequence of this invention beingexpressed does not begin with a methionine, e.g., a mature PI-linkedform of LFA-3 which begins with a phenylalanine, the start signal willresult in an additional amino acid --methionine --being located at theN-terminus of the product. While such methionyl-containing-product maybe employed directly in the compositions and methods of this invention,it is usually more desirable to remove the methionine before use.Methods are available in the art to remove such N-terminal methioninesfrom polypeptides expressed with them. For example, certain hosts andfermentation conditions permit removal of substantially all of theN-terminal methionine in vivo. Other hosts require in vitro removal ofthe N-terminal methionine. However, such in vivo and in vitro methodsare well known in the art. Furthermore, the LFA-3's of this inventionmay include amino acids in addition to the N-terminal methionine at theN-terminus. The LFA-3 may be used with those amino acids or they may becleaved with the N-terminal methionine before use.

A wide variety of host/expression vector combinations may be employed inexpressing the DNA sequences of this invention. Useful expressionvectors, for example, may consist of segments of chromosomal,non-chromosomal and synthetic DNA sequences, such as various knownderivatives of SV40 and known bacterial plasmids, e.g., plasmids from E.coli including col El, pCRl, pBR322, pMB9 and their derivatives, widerhost range plasmids, e.g., RP4, phage DNAs, e.g., the numerousderivatives of phage λ, e.g., NM989, and other DNA phages, e.g., M13 andFilamenteous single stranded DNA phages, yeast plasmids such as the 2μplasmid or derivatives thereof, vectors useful in eukaryotic cells, suchas vectors useful in animal cells and vectors derived from combinationsof plasmids and phage DNAs, such as plasmids which have been modified toemploy phage DNA or other expression control sequences. In the preferredembodiments of this invention, we employ PBG368,a pBG312-related vector[R. Cate et al., Cell, 45, pp. 685-98(1986)].

In addition, any of a wide variety of expression control sequences--sequences that control the expression of a DNA sequence whenoperatively linked to it --are used in these vectors to express the DNAsequences of this invention. Such useful expression control sequences,include, for example, the early and late promoters of SV40 or theadenovirus, the lac system, the trp system, the TAC or TRC system, themajor operator and promoter regions of phage λ, the control regions offd coat portein, the promoter for 3-phosphoglycerate kinase or otherglycolytic enzymes, the promoters of acid phosphatase, e.g., Pho5, thepromoters of the yeast α-mating factors, and other sequences known tocontrol the expression of genes of prokaryotic or eukaryotic cells ortheir viruses, and various combinations thereof. For animal cellexpression (e.g., L-M (tk⁻) cells), we prefer to use an expressioncontrol sequence derived from the major late promoter of adenovirus 2.

A wide variety of unicellular host cells are also useful in expressingthe DNA sequences of this invention. These hosts may include well knowneukaryotic and prokaryotic hosts, such as strains of E. coli,Pseudomonas, Bacillus, Streptomyces, fungi, such as yeasts, and animalcells, such as CHO and R1.1, B-W and L-M cells, African green monkeycells, such as COSl, COS7, BSCl, BSC40, and BMTl0, and human cells andplant cells in tissue culture. For expression of a soluble form ofLFA-3, an appropriate host cell is defective in the PI attachment ofproteins. We prefer L-M (tk⁻) cells.

It should of course be understood that not all vectors, expressioncontrol sequences and hosts will function equally well to express theDNA sequences of this invention. Neither will all hosts function equallywell with the same expression system. However, one of skill in the artmay make a selection among these vectors, expression control sequences,and hosts without undue experimentation and without departing from thescope of this invention. For example, in selecting a vector, the hostmust be considered because the vector must replicate in it. The vector'scopy number, the ability to control that copy number, and the expressionof any other proteins encoded by the vector, such as antibiotic markers,should also be considered.

In selecting an expression control sequence, a variety of factors shouldalso be considered. These include, for example, the relative strength ofthe system, its controllability, and its compatibility with theparticular DNA sequence, of this invention, particularly as regardspotential secondary structures. Unicellular hosts should be selected byconsideration of their compatibility with the chosen vector, thetoxicity of the product coded on expression by the DNA sequences of thisinvention to them, their secretion characteristics, their ability tofold proteins correctly, their fermentation requirements, and the easeof purification of the products coded on expression by the DNA sequencesof this invention.

Within these parameters one of skill in the art may select variousvector/expression control system/host combinations that will express theDNA sequences of this invention on fermentation or in large scale animalculture, e.g., mouse cells or CHO cells.

The polypeptides produced on expression of the DNA sequences of thisinvention may be isolated from the fermentation or animal cell culturesand purified in a variety of ways well known in the art. Such isolationand purification techniques depend on a variety of factors, such as howthe product is produced, whether or not it is soluble or insoluble, andwhether or not it is secreted from the cell or must be isolated bybreaking the cell. One of skill in the art, however, may select the mostappropriate isolation and purification techniques without departing fromthe scope of this invention.

The polypeptides of this invention are useful in compositions andmethods to block or to augment the immune responses. For example, theyare active in inhibiting cytolytic T-lymphocyte activity by interferingwith T-cell interaction with target cells. They have a similar blockingor augmenting effect on the immune response because they interfere withthe interaction of helper T-cells and target cells. Furthermore, thecompounds of this invention may be used to target specific T cells forlysis and immune suppression or to deliver drugs, such as lymphokines,to the specifically targeted T-cells. More preferably, solublederivatives of the polypeptides of this invention may be employed tosaturate the CD2 sites of T-lymphocytes thus inhibiting T-cellactivation. This is plainly of great utility in graft-vs-host disease,in autoimmune diseases, e.g., rheumatoid arthritis, and in preventingallograft rejection. Furthermore, the polypeptides of this invention arepreferred over monoclonal antibodies to a PI-linked form of LFA-3 or CD2because the polypeptides of this invention are less likely to elicitimmune responses in humans than are antibodies raised in species otherthan humans. The therapeutic compositions of this invention typicallycomprise an immunosuppressant or enhancement effective amount of suchpolypeptide and a pharmaceutically acceptable carrier. The therapeuticmethods of this invention comprise the steps of treating patients in apharmaceutically acceptable manner with those compositions.

The compositions of this invention for use in these therapies may be ina variety of forms. These include, for example, solid, semi-solid andliquid dosage forms, such as tablets, pills, powders, liquid solutionsor suspensions, liposomes, suppositories, injectable and infusablesolutions. The preferred form depends on the intended mode ofadministration and therapeutic application. The compositions alsopreferably include conventional pharmaceutically acceptable carriers andadjuvants which are known to those of skill in the art. Preferably, thecompositions of the invention are in the form of a unit dose and willusually be administered to the patient one or more times a day.

Generally, the pharmaceutical compositions of the present invention maybe formulated and administered using methods and compositions similar tothose used for other pharmaceutically important polypeptides (e.g.,alpha-interferon). Thus, the polypeptides may be stored in lyophilizedform, reconstituted with sterile water just prior to administration, andadministered by the usual routes of administration such as parenteral,subcutaneous, intravenous or intralesional routes.

The polypeptides of this invention or antibodies against them are alsouseful in diagnostic compositions and methods to detect T-cell subsetsor CD2+cells or to monitor the course of diseases characterized byexcess or depleted T-cells, such as autoimmune diseases, graft versushost diseases and allograft rejection. Still further, the polypeptidesof this invention may be used to screen for inhibitors of LFA-3 mediatedadhesion useful for inhibiting activation of T lymphocytes or Tlymphocyte mediated killing of target cells. Such screening techniquesare well-known in the art.

Finally, the polypeptides of this invention or antibodies against themare useful in separating B and T cells. For example, when bound to asolid support the polypeptides of this invention or antibodies to themwill separate B and T cells.

In order that this invention may be better understood, the followingexamples are set forth. These examples are for purposes of illustrationonly, and are not to be construed as limiting the scope of the inventionin any manner.

Synthesis Of Oligonucleotide Probes

We obtained a sample of LFA-3 (Dana Farber Cancer Institute, Boston,Massachusetts) previously purified as described by M. Dustin et al., J.Exp. Med., supra and sequenced as described by B. Wallner et al., supra.Next, we chemically synthesized two pools of anti-sense oligonucleotideDNA probes coding for regions from the amino terminal sequence of oursample of LFA-3 characterized by minimal nucleic acid degeneracy (seeunderscoring in FIG. 1) on an Applied Biosystems 30A DNA synthesizer.For each selected amino acid sequence, we synthesized pools of probescomplementary to all possible codons. We synthesized the probesanti-sense to enable hybridization of them to the correspondingsequences in DNA as well as in mRNA. We labelled our oligonucleotideprobes using -ATP and polynucleotide kinase (Maxam and Gilbert, Proc.Natl. Acad. Sci, USA, 74, p. 560 (1977)).

As depicted in FIG. 2, the oligonucleotide probe pool LF1 was a 20-merwith 32-fold degeneracy. Probe pool LF2-5, was a 20-mer with 384-folddegeneracy. However, to reduce its degeneracy, we synthesized this poolin four subpools of 96-fold degeneracy each by splitting the degeneratecodons for Gly into one of its four possible triplets for each subpool.We then selected the subpool containing the correct sequence from thethree pools containing incorrect sequences by hybridization of theindividual subpools to Northern blots containing human tonsil mRNA, asdescribed previously (Wallner et al., Nature, 320, pp. 77-81 (1986)).Oligonucleotide probe subpool LF2 hybridized to a 1300 nucleotidetranscript in human tonsil RNA, which suggested that it contained thecorrect sequence. Hence, we used it and pool LFl for screening ourvarious libraries.

Construction Of λgt10 Peripheral Blood Lymphocytes cDNA Library

To prepare our Peripheral Blood Lymphocytes (PBL) DNA library, weprocessed PBL from leukophoresis #9 through one round of absorption toremove monocytes. We then stimulated the non-adherent cells with IFN-γ1000 U/ml and 10 μg/ml PHA for 24 h. We isolated RNA from these cellsusing phenol extraction (Maniatis et al., Molecular Cloning, p. 187(Cold Spring Harbor Laboratory) (1982)) and prepared poly A⁺ mRNA by oneround of oligo dT cellulose chromatography. We ethanol precipitated theRNA, dried it in a SPEED-VAC ® vacuum centrifuge and resuspended the RNAin 10 [l H₂ O (0.5 μg/μl ). We treated the RNA for 10 min at roomtemperature in CH₃ HgOH (5mM final concentration) and β-mercaptoethanol(0.26 M). We then added the methyl mercury treated RNA to 0.1 M Tris-HCl(pH 8.3) at 43° C., 0.01 , MgCl₂ 0.01 M DTT, 2 mM Vanadyl complex, 5 μgoligo dT₁₂₋₁₈, 20 mM KCl, 1 mM dCTP, dGTP, dTTP, 0.5 mM dATP, 2 μCi[α-³²P]dATP and 30 U 1.5 μl AMV reverse transcriptase (Seikagaku America) ina total volume of 50 μl. We incubated the mixture for 3 min at roomtemperature and 3 h at 44° C. after which time we stopped the reactionby the addition of 2.5 μl of 0.5 M EDTA.

We extracted the reaction mixture with an equal volume ofphenol:chloroform (1:1) and precipitated the aqueous layer two timeswith 0.2 volume of 10 M NH₄ OAC , and 2.5 volumes EtOH and dried itunder vacuum. The yield of cDNA was 1.5 μg.

We synthesized the second strand according to the methods of Okayama andBerg (Mol. Cell. Biol., 2, p. 161 (1982)) and Gubler and Hoffman (Gene,25, p. 263 (1983)), except that we used the DNA polymerase I largefragment in the synthesis.

We blunt ended the double-stranded cDNA by resuspending the DNA in 80 μlTA buffer (0.033 M Tris Acetate (pH 7.8); 0.066 M KAcetate; 0.01MMgAcetate; 0.001M DTT; 50 μg/ml BSA), 5 μg RNase A, 4 units RNase H, 50μM α AND , 8 units E.coli ligase, 0.3125 mM dATP, dCTP, dGTP, and dTTP,12 units T₄ polymerase and incubated the reaction mixture for 90 min at37° C., added 1/20 volume of 0.5M EDTA, and extracted with phenol:chloroform. We chromatographed the aqueous layer on a cross-linkeddextran gel filtration column SEPHADEX G-150, Pharmacia, Piscataway, NJ)in 0.01M Tris-HCl (pH 7.5), 0.1 M NaCl, 0.001 M EDTA and collected thelead peak containing the double-stranded cDNA and ethanol precipitatedit. Yield: 605 μg cDNA.

We ligated the double-stranded cDNA to linker 35/36 ##STR1## usingstandard procedures. We then size selected the cDNA for 800 bp andlonger fragments on a gel filtration column of dextran cross-linked withacrylamide (SEPHACRYL S-500, Pharmacia, Piscataway, NJ) and ligated itto EcoRI digested λgtl0. We packaged aliquots of the ligation reactionusing a commercially available λ phage packaging extract (GIGAPAK,Stratagene, La Jolla, CA) according to the manufacturer's protocol. Weused the packaged phage to infect E.coli BNN102 cells and plated thecells for amplification. The resulting library contained 1.125×10⁶independent recombinants.

Screening Of The Libraries

We screened the PBL cDNA library prepared above with our labelledoligonucleotide probe LFl using the plaque hybridization screeningtechnique of Benton and Davis (Science, 196, p. 180 (1977)).

We pelleted an overnight culture of BNN102 cells in L broth and0.2maltose and resuspended it in an equal volume of SM buffer (50 mMTris-HCl (pH 7.5), 100 mM NaCl, 10 mM MgSO₄, and 0.01% gelatin).Thereafter, we preadsorbed 9 ml of cells with 1.5×10⁶ phage particles atroom temperature for 15 minutes and plated them on 30 LB Mg plates.

After incubation at 37° C. for 8 hours, we adsorbed plaques onto filtersfrom the plates and lysed the filters by placing them onto a pool of 0.5N NaOH/1.5 M NaCl for 5 minutes, and then submerged them for 5 min inthe same buffer. We neutralized the filters by submerging them in 0.5 MTris-HCl (pH 7.4), 1.5 M NaCl, two times for 5 minutes each, and rinsedthem for 2 minutes in 1 M NH₄ OAc, air dried the filters, and baked themfor 2 hours at 80° C.

We prehybridized and hybridized the filters to oligonuoleotide probe LFlin 0.2% polyvinylpyrolidone, 0.2% FICOLL (MW 400,000), 0.2% bovine serumalbumin, 0.05 M Tris-HCl (pH 7.5), 1 M sodium chloride, 0.1% sodiumpyrophosphate, 1% SDS, and 10% dextran sulfate (MW 500,000). We detectedthe hybridizing λ-cDNA sequences by autoradiography.

We initially selected 26 positive phages from the PBL library andrescreened these clones and plaque purified them at lower density usingthe same probe.

Sequencing Of The P24 cDNA Clone

We characterized the cDNA from a clone, P24, screened above by DNAsequencing analysis. We subcloned the NotI digested DNA from clone λP24into vector pNN01 to give p24 and to facilitate sequence analysis.* Theentire insert of λP24 is contained on a single NotI fragment. Forsubcloning, we used the vector's EcoRI site or SmaI site employingtechniques in common use.

We determined the DNA sequences of our subclones largely by the, methodof Maxam and Gilbert (Meth. Enzymology, 65 pp. 499-560) 1980). However,for some fragments, we used the related procedure of Church and Gilbert(Proc. Natl. Acad. Sci. USA, 81, p. 1991 (1984)). The structure of pNN01enables sequencing, by the Church-Gilbert approach, of the ends of aninserted fragment using NotI digestion and four 20-nucleotide longprobes: NN-A, NN-B, NN-C and NN-D. See FIG. 5.

FIG. 3 shows the DNA sequence of the cDNA insert of phage λP24. It alsodepicts the amino acid sequence deduced therefrom.

Determination Of Linkage Form Of LFA-3 From P24 cDNA

We decided to characterize the linkage form of LFA-3 coded for by P24cDNA. We choose the Rl.1 cell line because it is known to expresssurface antigens that are attached to the membrane by a PI linkage.

We incubated 5×10⁶ cells of clones P24/Rl.1, HT16/Rl.1 (an Rl.1 cellline transfected with cDNA coding for a membrane integrated form ofLFA-3 (see, B. Wallner et al., supra)) and Rl.1 cells with 0.5 μl ofphosphatidylinositol-specific phospholipase C (PIPLC) at 37° C. for 1hour. It is known that PIPLC upon incubation releases PI-linked proteinsfrom the cell surface, while it has no effect on proteins attached tothe cell surface by other mechanisms such as membrane integratedproteins (M. Low, Biochem. J., 244, p. 1 (1987)).

We determined the amount of LFA-3 released from the cell surface ofP24/Rl.1 or HT16/Rl.1 by the decrease of surface fluorescence assayed onFACS. We found that incubation of P24/Rl.1 cells with PIPLC resulted inthe release of 95% of surface LFA-3 while PIPLC did not have any effecton the fluorescence of R1.1 cells or HT16/R1.1 cells. This indicatesthat P24 cDNA codes for the PI-linked form of LFA-3.

Adhesion Of a PI-linked form of LFA-3 From P24/R1.1 To Other Cells

We next tested whether a PI-linked form of LFA-3 from P24 cDNA asexpressed in R1.1 cells would mediate adherence of P24/R1.1 to othercells. We tested this by rosetting analysis with L-cells expressing CD2cDNA (L114). We grew control L cells and L114 (CD2 transfected) cells,in a 9.6 cm² well of a 6 well tissue culture plate at a cell density of3×10⁵ cells per well. After washing the wells twice with Roswell ParkMemorial Institute (RPMI 1060) medium to remove cell debris and deadcells, 1.5 ×10⁷ P24/R1.1 or R1.1 cells as a control were added per well.Plates were spun at 400 rpm for 2 minutes in a Sorvall Centrifuge at 4°C. After the cells were incubated at 4° C. for 2 hours, the wells werewashed with RPMI 1060 medium to remove excess P24/R1.1 or R1.1 cells.P24/R1.1 cells rosetted with the L114 cells as determined under themicroscope. We observed rosetting of P24/R1.1 with L114 cells but notwith the untransfected control cells. This rosetting could be inhibitedwith MAb to LFA-3 (TS2/9) or MAb to CD2 (TS2/18). This indicates that aPI-linked form of LFA-3 is expressed on cell surface of R1.1 cells in aconformation that allows interaction with recombinant CD2 expressed onmouse L-cells. P24/R1.1 cells or untransfected R1.1 cells do not rosettewith untransfected mouse L-cells, indicating the specificity of thesecellular interactions.

Expression Of PI-linked Form Of LFA-3 From P24 cDNA In CHO cells

We inserted a Klenow blunt-ended NotI PI-linked form of LFA-3 cDNAfragment of p24 into a blunt-ended SalI site of plasmid pJOD-s to givepJOD-s-LFA3P24.

Vector pJOD-s has been deposited in the In Vitro International, Inc.Culture Collection, 611 P. Hammonds Ferry Rd., Linthicum, Maryland,21090 on July 22, 1988 and has been assigned accession number 10179.This deposit was transferred to the American Type Culture collection,Rockville, Maryland, on Jun. 20, 1991 and assigned accession number ATCC68787.

We next linearized pJOD-s-LFA3P24 with PvuI for transfection of CHOcells. We incubated 10μg of PvuI linearized DNA with 0.125 M CaCl₂ in TEand 1×HEBS (137 mM NaCl, 5mM KCl, 0.0030 M Na₂ HPO₄, 0.7 H₂ O, 6mMDextrose, 20 mM Hepes (pH 7.1)) at room temperature for 20 minutes. DNAwas added to cells in alpha⁺ -MEM medium and incubated at 37° C. for 4hours. After removing the medium, cells were incubated at roomtemperature for 4 minutes in alpha⁺ -MEM +10% glycerol. Cells wererinsed with medium and grown for 2 days in alpha⁺ -MEM, then transferredto selective medium (alpha⁻ -MEM).

We determined expression of a PI-linked form of LFA-3 by FACS analysis.To analyze by FACS, 1×10⁶ cells per each P24-CHO methotrexate clone andcontrol CHO cells were removed from the tissue culture dishes byincubation with Hank's BSS buffer, 0.5 M EDTA at 4° C. for 15 minutes.The detached cells were then pelleted, resuspended in 50 μl of PBNbuffer (1×PBS,0.5% BSA, 0.1% sodium azide) and incubated with 100 μl ofMAb TS2/9 (1.2 mg/ml) (a gift of Tim Springer) on ice for 45 minutes. Wenext washed the cells two times with 1 ml PBN buffer and pelleted bycentrifugation. The cell pellets were resuspended in 100μl of a 1:50dilution of FCI (Fluorescein Conjugated Affinity Purified F (ab')₂Fragment Sheep Anti-Mouse IgG (Cappel, Biomedical, Pennsylvania)) in PBNbuffer and incubated on ice for 30 minutes. Cells were pelleted bycentrifugation and excess FCI was removed by resuspending the cellpellets twice in 1 ml PBN buffer. We then resuspended the cells in 800μl of 1×PBS and determined the fluorescence intensity on FACS. Weobserved five clones showed between 5 to 50 fold higher fluorescencethan control CHO cells.

Expression Of A PI-linked Form Of LFA-3 From P24 cDNA In R1.1 cells

We used expression vector pBG24 derived from expression vector pBG312.pBG24 was constructed by digesting plasmid p24 DNA with NotI andblunt-ended with Klenow. We next isolated a 860 bp NotI fragment of p24followed by ligation with an EcoRI linearized, blunt-ended expressionvector PBG368. PBG368 was constructed as follows. Animal expressionvector pBG312 (described R. Cate et al., Cell, 45, pp. 685-98 (1986) wasdigested with EcoRI and BglII to delete one of each of the two EcoRI andthe two BglII restriction sites (the EcoRI site at position 0 and theBglII site located at approximately position 900). A bacterial strain(pBG312.hmis/JA221) harboring plasmid pBG312 bearing a DNA insert codingfor human Mullerian inhibiting substance was deposited with In VitroInternational, Inc., Linthicum, Maryland, on Oct. 23, 1985, assignedaccession number IVI-10089 and has been publicly available since Sep.10. 1991. This deposit was transferred to the American Type CultureCollection, Rockville, Maryland on Jun. 20, 1991, an dis available fromthere under accession number ATCC 68813.

90 μg DNA of pBG24 was linearized with NruI, and cotransfected with 10μg of NruI linearized pTCF DNA (F. Grosveld et al., Nucleic Acid Res.,10, p. 6715 (1982)) and 300 μg sonicated salmon sperm DNA by DNAelectroporation using a BIORAD (Richmond, California) GENE PULSER ® setat 0.29 kV with capacitance set at 960 μFd. We selected for transfectionR1.1 cells in RPMI 1060 medium +1 mg/ml G418. We selected single clonesafter limiting dilutions to 10³ cells per well in a 96 well dish inselective medium. Eight clones, resistant to G418, were assayed for aPI-linked form of LFA-3 expression by FACS analysis as described above.All eight P24/R1.1 clones expressed PI-linked form of LFA-3 at a level10 to 1000 fold above R1.1 control cells.

Expression Of A PI-linked Form Of LFA-3 P24 cDNA In L Cells

To express our P24 cDNA in mouse L cells, we cotransfected 90 μg ofplasmid pBG24 DNA ,as described above, that was linearized with NruIwith 10μg plasmid pOPF DNA carrying a thymidine kinase gene (tk)(Grosveld et al., supra), linearized with ScaI into 1×10⁷ L-M (tk-)cells (C. P. Terhorst, J. Immun., 131, p. 2032 (1983)) byelectroporation as described above. We selected for transfected cells bytk expression by growing them in DMEM +HAT at cell densities of 1×10⁵cells per 100 mm plate. Clones were picked and expanded to 5×10⁵ cellsper 100 mm dish to assay for expression of a PI-linked form of LFA-3 byFACS analysis as described above. We observed some expression at levelsabove control cells, although 70% of the PI-linked form of LFA-3 wassecreted into the medium as discussed below.

Secretion Of LFA-3 From P24/L Cells

We further wanted to test whether P24/L cells secrete LFA-3 because thismouse L cell line--L-M(tk⁻) --is known to be deficient in a PI linkageattachment mechanism. P24/L cells were metabolically labeled with ³⁵S-met and the 35S-labelled PI-linked form of LFA-3 was precipitated fromthe medium with MAb TS2/9 (a gift of Tim Springer) as follows. 3 ×10⁵P24/L, HT16/L (B. Wallner et al., supra) or L(tk-) cells were plated in1 well each of a 6 well cell culture plate, grown overnight in DMEM-HATcomplete medium (DMEM +HAT +10% FCS +glutamine). Wells were then rinsedwith lx Minimal Essential Medium Eagle (modified) methionine free (MEM).For ³⁵ S met labeling, we added 1.5 ml of MEM medium (methionine free),plus glutamine, 2.5% complete DMEM and 225 μCi ³⁵ S met (New EnglandNuclear, Delaware, 1135 mCi/μm) to each well and incubated at 37° C. for18 hours. To 0.7 ml of medium 10 μl of MAb TS2/9 coupled to agarose wasadded, and the mixture rocked at 4° C. overnight. To each well we added300 μl of DOC buffer (20 mM Tris (pH 7.3), 50mM sodium chloride, 0.5%deoxycholate, 0.5% cells off the plates, transferred to Eppendorf tubes,vortexed and centrifuged for 15 minutes at room temperature. To 100 μlof the supernatant, 10 μl of MAb TS2/9 coupled to agarose was added andincubated overnight at 4° C. with rocking. The TS2/9-agarose ³⁵ S-LFA-3complex was pelleted by centrifugation, washed three times with 1 ml ofDOC buffer, and resuspended in 50 μl SDS-loading buffer. ³⁵ S-LFA-3(PI-linked form) was dissociated from TS2/9-agarose by heating thecomplex to 65° C for 10 minutes. The TS2/9 agarose was precipitated bycentrifugation and 25 μl of the supernatant was electrophoresed on areducing SDS-polyacrylamide gel. We observed precipitation of the 55 kd³⁵ S-labelled protein with MAb TS2/9 only from the medium of P24/L cellsand not from the medium of L-M(tk⁻) control cells.

We determined by SDS-PAGE that the ³⁵ S-LFA-3 labeled LFA-3, secretedfrom P24/L cells is approximately 3 kd smaller than the ³⁵ S-labelledLFA-3 retained intracellularly in P24/L cells or HT16/L cells. Thisindicates that a portion or all of the hydrophobic potentialtransmembrane region is removed before secretion, which prevents theefficient integration of a PI-linked form of LFA-3 into the cell surfacemembrane.

We deposited the following plasmid carrying a PI-linked form of LFA-3cDNA sequence of this invention in the In Vitro International, Inc.Culture Collection in Linthicum, Maryland, on Jul. 22, 1988:

The plasmid has been assigned accession number IVI-10180. This depositwas transferred to the American Type Culture Collection, ockville,Maryland, on Jun. 20, 1991 and has been assigned accession number ATC68788.

While we have hereinbefore presented a number of embodiments of thisinvention, it is apparent that our basic construction can be altered toprovide other embodiments which utilize the processes and compositionsof this invention. Therefore, it will be appreciated that the scope ofthis invention is to be defined by the claims appended hereto ratherthan the specific embodiments which we have presented by way of example.

We claim:
 1. A DNA molecule with a DNA sequence selected from the groupconsisting of:(a) the DNA sequence of the DNA insert carried in phageλP24; and (b) DNA sequences which are degenerate to the foregoing DNAsequence.
 2. A DNA molecule with a DNA sequence selected from the groupconsisting of a DNA sequence of the formula N₁₋₈₃₀ of FIG. 3, a DNAsequence of the formula N₁₈₋₈₃₀ of FIG. 3, a DNA sequence of the formulaN₁₀₂₋₈₃₀ of FIG. 3 and DNA sequences which are degenerate to any of theforegoing DNA sequences.
 3. A DNA molecule with a DNA sequence selectedfrom the group consisting of a DNA sequence of the formula N₁₋₆₅₃-N₇₃₈₋₈₃₀ of FIG. 3, a DNA sequence of the formula N₁₀₂₋₆₅₃ -N₇₃₈₋₈₃₀ ofFIG. 3, a DNA sequence of the formula N₁₋₆₆₂ -N₇₃₈₋₈₃₀ of FIG. 3, a DNAsequence of the formula N₁₀₂₋₆₆₂ -N₇₃₈₋₈₃₀ of FIG. 3, a DNA sequence ofthe formula N₁₋₆₃₈ -N₇₃₈₋₈₃₀ of FIG. 3, a DNA sequence of the formulaN₁₀₂₋₆₃₈ -N₇₃₈₋₈₃₀ of FIG. 3, a DNA sequence of the formula N₁₋₇₀₁-N₇₃₈₋₈₃₀ of FIG. 3, a DNA sequence of the formula N₁₀₂₋₇₀₁ -N₇₃₈₋₈₃₀ ofFIG. 3, and DNA sequences that are degenerate to any of the foregoingDNA sequences.
 4. A recombinant DNA molecule comprising an isolated DNAmolecule according to claim 1 or 2, said DNA molecule being operativelylinked to an expression control sequence in said recombinant DNAmolecule.
 5. A recombinant DNA molecule comprising a DNA molecule ofclaim 3, said DNA molecule being operatively linked to an expressioncontrol sequence in said recombinant DNA molecule.
 6. The recombinantDNA molecule according to claim 4, wherein the molecule ispJOD-s-LFA3P24.
 7. A unicellular host transformed with a recombinant DNAmolecule according to claim
 4. 8. A unicellular host transformed with arecombinant DNA molecule according to claim
 5. 9. A unicellular hosttransformed with a recombinant DNA molecule according to claim 4 whereinsaid host is selected from the group consisting of CHO cells and R1.1cells.
 10. A unicellular host transformed with a recombinant DNAmolecule according to claim 4 wherein said host is L-M(tk⁻).
 11. Therecombinant DNA molecule according to claim 4, wherein side expressioncontrol sequence is selected from the group consisting of the early orlate promoters of SV 40 or adenovirus, the lac system, the trp system,the TAC system, the TRC system, the major operator and promoter regionsof phage λ, the control regions of fd coat protein, the promoter for3-phosphoglycerate kinase or other gylcolytic enzymes, the promoters ofacid phosphatase and the promoters of the yeast α-mating factors. 12.The recombinant DNA molecule according to claim 5, wherein saidexpression control sequence is selected from the group consisting of theearly or late promoters of SV40 or adenvirus, the lac system, the trpsystem, the TAC system, the TRC system, the major operator and promoterregions of pahge λ, the control regions of fd coat protein, the promoterfor 3-phosphoglycerate kinase or other glycolytic enzymes, the promotersof acid phosphatase and the promoters of the yeast α-mating factors. 13.A unicellular host transformed with a recombinant DNA molecule accordingto claim 4, wherein said host is selected from the group consisting ofstrains of E.coli, Pseudomonas, Bacillus, Streptomyces, yeast, fungi,animal cells, plant cells and human cells in tissue culture.
 14. Aunicellular host transformed with a recombinant DNA molecule accordingto claim 4, wherein said unicellular host secretes a soluble from ofLFA-3 when transformed with the DNA insert carried in phage λP24.
 15. Aunicellar host transformed with a recombinant DNA molecule according toclaim 4, wherein cell surface proteins of said unicellular host areresistant to phosphatidylinositol-specific phospholipase C treatment.